Microsample treatment apparatus

ABSTRACT

The present invention aims at providing a structure whereby, in the step of injecting a sample in a microquantity into a well, the migration of the sample into another well or overflow thereof can be prevented; and a structure whereby the position of the injected sample can be adjusted in a well or the sample can be transferred into the next well under controlling. It also aims at providing an apparatus for detecting chemotaxis of cells and separating chemotactic cells with the application of the above structures.  
     Accordingly, the present invention provides a microsample treatment apparatus and an apparatus for detecting chemotaxis of cells and separating chemotactic cells wherein a plural number of wells are connected to each other via a part having resistance to fluids and the wells are each provided with tubes for injecting/sucking a sample and, if necessary, tubes for relieving pressure changes at the injection/suction, characterized in that these tubes have a space in common at the top ends thereof in which a liquid can be held.

TECHNICAL FIELD

[0001] This invention relates to an apparatus for treating liquidsamples in microquantities. More particularly, it relates to amicrosample treatment apparatus having a structure whereby, in the stepof injecting a liquid sample into a microwell for holding a sample to bereacted, analyzed, detected, etc., the overflow of the sample or themigration thereof into another well connected thereto can be preventedand the position of the sample in the microwell can be adjusted.

[0002] The present invention further relates to an apparatus for judgingwhether or not cells can migrate in a definite direction by their ownactions, observing the state of cells migrating in a definite directionby their own actions, or counting cells having migrated in a definitedirection by their own actions (i.e., an apparatus for detectingchemotaxis of cells). Furthermore, the present invention relates to anapparatus for separating cells based on the selective migration of cellsby their own actions. More particularly speaking, it relates to anapparatus for detecting chemotaxis of cells or separating chemotacticcells having a structure wherein, in the step of injecting a liquidsample into a microwell for holding a cell suspension or aspecimen/sample to be detected, separated, etc., the overflow of thesample or the migration thereof into another well connected thereto canbe prevented and the position of the sample in the microwell can beadjusted.

BACKGROUND ART

[0003] With the recent development and progress in nanotechnology, ithas been apractice to handle cells, proteins, genes and so on at a levelof several individuals. As a result, it becomes necessary to inject andtreat microsamples into containers (wells) for reaction, analysis ordetection. To carry out a series of reactions, analyses, detections,etc. on microchips, use is sometimes made of a structure wherein aplural number of wells are connected to each other each via a pipe, agroove or a channel. In such a case, attention should be taken toprevent the migration of a sample into the adjacent well due to theinjection pressure, which brings about some difficulties not only inmanual operations but also in operations with the use of an automaticinjection device. It is also desired to adjust the position of a sampleinjected into a microwell or to transfer the sample into the next wellwhile adjusting the position.

[0004] It is an object of the present invention to provide a structureto be used in the above-described apparatus whereby, in the step ofinjecting amicrosample into a well, the migration of the sample intoanother well or overflow from the well can be certainly prevented. It isanother object of the present invention to provide a structure whereinthe position of an injected sample in a well can be adjusted or thesample can be transferred into the next well under controlling. It isstill another object of the present invention to provide a microsampletreatment apparatus wherein a sample can be injected and transferredunder automated control.

[0005] It is still another object of the present invention to provide anapparatus for detecting chemotaxis of cells or separating chemotacticcells with the application of the structure having the functions asdescribed above.

DISCLOSURE OF THE INVENTION

[0006] The present invention relates to a microsample treatmentapparatus having a structure wherein a plural number of wells areconnected to each other via a part having resistance to fluids and thewells are each provided with tubes for injecting/sucking a sample and,if necessary, tubes for relieving pressure changes at theinjection/suction, characterized in that these tubes have a space incommon at the top ends thereof in which a liquid can be held. The parthaving resistance to fluids may be selected from among one or more thinpipes, narrow gaps, thin grooves, filters, resin-filled columns andother structures through which a fluid can be passed but which haveresistance to fluids.

[0007] The present invention further relates to a microsample treatmentapparatus wherein the top end of a tube formed in a well is locatedupper than the top ends of the tubes formed in one or more wellsopposite thereto across the part having resistance to fluids. Themicrosample treatment apparatus according to the present invention mayhave, in one or both of wells connected to each other via a channel, awall formed orthogonal to the channel to thereby restrict the amount ofa liquid in the vicinity of the channel.

[0008] The present invention relates to a microsample treatmentapparatus which comprises a unit part having a single unit selected fromthe microsample treatment apparatuses as described above, an integrationunit having a plural number of units of the same or different types or aplural number of integration units, a pipette or pipettes forcontrolling the liquid level in the unit part, and a system forcontrolling the operation of the liquid level control pipette(s).Moreover, the present invention relates to an automated microsampletreatment apparatus characterized in that the liquid level controlpipette(s) are controlled so as to suck a definite amount of a liquidcontained in the space held in common by a plural number of tubes at thetop ends thereof in each of the units in the unit part, therebyadjusting the position of the sample in well(s) or transferring thesample into the respective next well(s) followed by, if necessary,supplying the liquid in a compensatory amount to return the liquid faceto the original level. If necessary, the microsample treatment apparatusmay be provided with a sample reservoir, a specimen reservoir,pipette(s) washing part and sample supply pipette(s) and specimen supplypipette(s) which are movable over these parts and further have a systemfor controlling the operations of these pipettes. The materials of thepipettes are not restricted to glass but can be appropriately selectedfrom among metals, plastics and the like.

[0009] The present invention involves in its scope an apparatus fordetecting chemotaxis of cells or separating chemotactic cellscharacterized in that a plural number of wells are connected to eachother via a part having resistance to fluids, the wells are eachprovided with tubes for injecting/sucking a sample and, if necessary,tubes for relieving pressure changes at the injection/suction, thesetubes have a space in common at the top ends thereof in which a liquidcan be held, and the wells are closely adhered to a glass substrate inthe side opposite to the tube side.

[0010] The present invention further relates to an apparatus fordetecting chemotaxis of cells or separating chemotactic cells asdescribed above characterized in that the top end of a tube formed in awell for holding cells is located upper than the top ends of the tubesformed in one or more wells opposite thereto across the channel havingresistance to fluids.

[0011] In the present invention, it is preferable that the channelhaving resistance to fluids is a bank and a narrow gap is formed betweenthe bank and the glass substrate. In this case, a terrace may be formedin the upper part of the bank in the channel to form a gap between theterrace and the glass substrate. Alternatively, barriers constitutingone or more grooves having a width fit for the diameter or deformabilityof cells may be formed in the upper part of the bank and, if necessary,a terrace may be further formed together with the bank to form a gap fitfor the diameter or deformability of cells between the terrace and theglass substrate too. A plural number of grooves in the direction towardthe opposite well in the channel may be connected to each other via oneor more grooves orthogonal thereto. It is also possible that the widthof a plural number of grooves in the direction toward the opposite wellin the channel is changed stepwise each time the grooves intersect oneor more grooves orthogonal thereto. Furthermore, a plural number ofgrooves in the direction toward the opposite well in the channel may beformed by mutually shifting the positions thereof each time the groovesintersect one or more grooves orthogonal thereto. Moreover, arrays ofthe barriers constituting the grooves may be formed at two positions inboth sides of the terrace formed at the center of the bank. It is alsopossible that multistage terraces are formed on the bank in the channelso as to form gaps with different depths between the terrace and theglass substrate. In one or both of wells connected to each other via achannel, moreover, a wall may be formed orthogonal to the channel tothereby restrict the amount of a liquid in the vicinity of the channel.

[0012] The present invention relates to an automated apparatus fordetecting chemotaxis of cells or separating chemotactic cells comprisinga unit part having a single unit selected from the apparatuses fordetecting chemotaxis of cells or separating chemotactic cells asdescribed above, an integration unit having a plural number of units ofthe same or different types or a plural number of integration units, acell reservoir, a specimen reservoir and liquid level controlpipette(s), cell supply pipette(s) and specimen supply pipette(s) whichare movable over these parts, and further having a detection part fordetecting cell migration in the unit part and, if necessary, recordingthe detection data which is integrated with the unit part or formed soas to correspond to a plural number of unit parts, and further having asystem for controlling the movements of the liquid level controlpipette(s), the cell supply pipette(s) and the specimen supplypipette(s) and, if necessary, a system for moving the unit part to thedetection part and the next unit part to the pipette flow line. Ifnecessary, this apparatus may further have a pipette washing part.

[0013] The present invention further relates to an automated apparatusfor detecting chemotaxis of cells or separating chemotactic cellscharacterized in that the operations of the respective pipettes arecontrolled as follows: after optionally stirring, a definite amount acell suspension is sucked by the cell supply pipette(s) and suppliedinto the unit part; then a definite amount of a liquid, which iscontained in the space held by the top ends of a plural number of tubesin common in each unit, is sucked by the liquid level control pipette(s)to thereby adjust the position of the cells in the wells; the liquid inthe compensatory amount is supplied from the liquid level controlpipette(s) into the space to thereby return the liquid face to theoriginal level; then a definite amount of a specimen is sucked from thespecimen reservoir by the specimen supply pipette(s) and supplied intothe unit part; then the pipettes move toward the pipette washing part inwhich they are washed by repeatedly sucking and discharging the washingliquor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a model view which shows an example of an apparatus fordetecting chemotaxis of cells or separating chemotactic cells previouslyproposed by the present inventors.

[0015]FIG. 2 is a bottom plan view of the apparatus of FIG. 1.

[0016]FIG. 3 is a model view which shows an example of the applicationof the structure according to the present invention to an apparatus fordetecting chemotaxis of cells or separating chemotactic cells. The arrowshows the liquid level of a liquid filling up the apparatus.

[0017]FIG. 4 is a model view showing another example of the applicationof the structure according to the present invention to an apparatus fordetecting chemotaxis of cells or separating chemotactic cells which isprovided with tubes 3 for injecting/collecting a sample into wells andtubes 4 for relieving decrease/increase in pressure at the step ofinjecting/collecting the sample. The arrow shows the liquid level of aliquid filling up the apparatus.

[0018]FIG. 5 is a model view showing another example of the applicationof the structure according to the present invention to an apparatus fordetecting chemotaxis of cells or separating chemotactic cells whereinthe top end 3Ab of a tube 3A in a well 2A for holding cells is locatedupper than the top end 3Bb of a tube 3B in another well 2B. The arrows Iand II show the liquid levels of a liquid filling up the apparatus.

[0019]FIG. 6 is a model view showing another example of the applicationof the structure according to the present invention to an apparatus fordetecting chemotaxis of cells or separating chemotactic cells which isprovided with tubes 3 for injecting/collecting a sample into wells andtubes 4 for relieving decrease/increase in pressure at the step ofinjecting/collecting the sample, wherein the top ends 3Ab and 4Ab oftubes 3A and 4A in a well 2A for holding cells are located upper thanthe top ends 3Bb and 4Bb of tubes 3B and 4B in another well 2B. Thearrows I and II show the liquid levels of a liquid filling up theapparatus.

[0020]FIG. 7 shows a modification example of the structure as shown byFIG. 6. The arrows I and II show the liquid levels of a liquid fillingup the apparatus.

[0021]FIG. 8 is a top plan view of a substrate in an example whereinwells are connected each via a channel in the triple system.

[0022]FIG. 9 is a top plan view of a substrate in an example wherein aplural number of wells 2B₁₋₄ are connected to a single well 2A each viaa channel 1.

[0023]FIG. 10 is a sectional view of an apparatus having the substrateas shown by FIG. 9 along the dashed dotted line in FIG. 9. The arrows Iand II show the liquid levels of a liquid filling up the apparatus.

[0024]FIG. 11 is a top plan view of an example wherein the connectionsystem in FIG. 9 is provided circularly.

[0025]FIG. 12 shows an example of the structure of a channel 1.

[0026]FIG. 13 shows an example of the arrangement of barriers 12 andgrooves 13 in a channel 1. The arrow shows the direction toward theopposite well.

[0027]FIG. 14 is a sectional view of the channel 1 shown by FIG. 13.

[0028]FIG. 15 shows an example wherein grooves 13 in the directiontoward the opposite well across a channel 1 are connected via twogrooves 14 orthogonal thereto. The arrow shows the direction toward theopposite well.

[0029]FIG. 16 shows an example of an integration of multiplicity ofunits wherein the units are all in the same type.

[0030]FIG. 17 shows an example of an integration of multiplicity ofunits wherein the units are in different types.

[0031]FIG. 18 shows an example wherein multiplicity of units arecircularly integrated.

[0032]FIG. 19 is a sectional view along the dashed dotted line in FIG.18.

[0033]FIG. 20 shows an example of the fabrication of an apparatus fordetecting chemotaxis of cells or separating chemotactic cells wherein(1) is perspective views of individual parts and (2) is sectional viewscorresponding thereto.

[0034]FIG. 21 is a model view of an apparatus wherein a well to bereacted and another well for holding the target substance are connectedvia a column. The arrows I and II show the liquid levels of a liquidfilling up the apparatus.

[0035]FIG. 22 is a model view of an apparatus for separating substances.The arrows I and II show the liquid levels of a liquid filling up theapparatus.

[0036]FIG. 23 shows an example wherein a bank 8 in a channel 1 hasmultistage terraces 11 ⁻¹⁻⁴.

[0037]FIG. 24 shows an example of wells wherein walls are formed along achannel.

[0038]FIG. 25 shows another example of wells wherein walls are formedalong a channel.

[0039]FIG. 26 shows an arrangement example wherein the wells shown byFIG. 24 are integrated.

[0040]FIG. 27 shows an example of an automatic controlling system of theapparatus according to the present invention.

[0041]FIG. 28 shows the movement of liquid level control pipette(s).

[0042]FIG. 29 shows an example of containers ina cell reservoir.

[0043]FIG. 30 shows an example of a container in a specimen reservoir.

[0044]FIG. 31 shows an arrangement example of the containers shown byFIG. 30 in the specimen reservoir.

[0045]FIG. 32 shows another example of a container in the specimenreservoir.

[0046]FIG. 33 shows an arrangement example of the containers shown byFIG. 32 in the specimen reservoir.

[0047]FIG. 34 shows an example of a pipette to be used in the presentinvention.

[0048]FIG. 35 shows an example wherein pipette tip inlets are formed inthe upper part of tubes for injecting/collecting a sample.

[0049]FIG. 36 shows an example wherein grooves in the direction towardthe opposite well across a channel are connected to each other via twogrooves formed orthogonally thereto and the width of the grooves in thedirection toward the opposite well is changed stepwise each time thegrooves intersect the grooves orthogonal thereto. Each arrow shows thedirection toward the opposite well. In this figure, the width of thebarriers per se is changed.

[0050]FIG. 37 shows an example of the modification of the structure ofFIG. 36 in which the barriers have the same size but are changed innumber. The arrow shows the direction toward the opposite well.

[0051]FIG. 38 shows an example wherein grooves in the direction towardthe opposite well across a channel are connected to each other via threegrooves formed orthogonally thereto and the grooves in the directiontoward the opposite well are formed by mutually shifting the positionsthereof each time the grooves intersect the grooves orthogonal thereto.In this figure, the grooves shift by ½ pitch toward the orthogonaldirection. Each arrow shows the direction toward the opposite well.

[0052]FIG. 39 shows an example wherein barriers are jointed in thedirection toward the opposite well. Each arrow shows the directiontoward the opposite well.

[0053]FIG. 40 shows an example wherein a terrace is formed at the centerof a bank and two arrays of barriers are formed in both sides of theterrace.

DESCRIPTION OF THE REFERENCE NUMERALS AND SIGNS

[0054] 1: channel.

[0055] 2: well. Appendixes A, B, B_(1-n), and C are provided todifferentiate the wells.

[0056] 3: tube for injecting/collecting samples. Appendixes A, B,B_(1-n), and C are provided to differentiate the wells. Appendix arepresents a penetrating hole corresponding to a tube 3 of a substrate5. Appendix b represents the top end of the tube 3.

[0057] 4: tube for avoiding increase/decrease in pressure atinjecting/collecting samples. Appendixes A, B, B_(1-n), and C areprovided to differentiate the wells. Appendix a represents a penetratinghole corresponding to a tube 4 of a substrate 5. Appendix b representsthe top end of the tube 4.

[0058] 5: substrate.

[0059] 5′: packing.

[0060] 6: glass substrate.

[0061] 7: block having tube mounted thereto.

[0062] 8: bank.

[0063] 9: detector.

[0064] 10: space held together by top ends of tubes.

[0065] 11, 11 _(−1 to 4): terraces.

[0066] 12: barrier in channel 1.

[0067] 13: groove in the direction toward the opposite well acrosschannel.

[0068] 14: groove formed orthogonally to groove 13.

[0069] 15: magnet.

[0070] 16: column located between wells.

[0071] 17: cover cap.

[0072] 18: O-ring.

[0073] 19: guide pin receiver hole.

[0074] 20: guide pin.

[0075] 21: intermediate base.

[0076] 22: bottom base.

[0077] 23: bottom substrate.

[0078] 24: wall formed along channel.

[0079] 25: cell reservoir.

[0080] 26: cell injection part.

[0081] 27: liquid injection part.

[0082] 28: specimen reservoir.

[0083] 29: pipette tip inlet port.

[0084] 30: pipette washing part.

[0085] 31: multichannel syringe.

[0086] 32: actuator.

[0087] 33: needle of automatic pipette.

[0088] 34: tip of manual pipette.

[0089] ←: level of liquid filling up apparatus.

[0090] ←I: level of liquid making the top end of upper tube submerged.

[0091] ←II: level of liquid making the top end of upper tube visibleabove the liquid face.

[0092] X-X′: flow line of specimen supply pipette.

[0093] Y-Y′: flow line of cell supply pipette.

[0094] Z-Z′: flow line of liquid level control pipette.

BEST MODE FOR CARRYING OUT THE INVENTION

[0095] The microsample treatment apparatus according to the presentinvention provided with wells into which a sample such as a liquid or asuspension is injected is an apparatus for handling organic or inorganicchemicals, polymers such as proteins, genes, cells and so on in thestate of solutions or suspensions. Although the structure of the presentinvention is not specifically restricted in the amount of samples to betreated, it is expected that high technical merits can be achievedthereby in case of using samples of the order of several milliliters tomicroliters.

[0096] The present invention is applied to case wherein a plural numberof wells are connected to each other via a structure having resistanceto fluids and the wells are each provided with tubes for injecting orsucking a sample and, if necessary, tubes for relieving pressure changesat the step of the injecting or sucking the sample. That is, such anapparatus has a plural number of tubes as a whole. In the presentinvention, these tubes have a space in common at the top ends thereof inwhich a liquid can be held. Owing to this structure, unexpectedmigration and overflow caused by a rapid change in pressure in the wellsin the step of injecting or sucking a sample or unexpected migration ofthe sample caused by horizontal off balance of the apparatus can beeffectively prevented.

[0097] By employing the structure wherein a plural number of tubes havea space in common at the top ends thereof in which a liquid can be held,the position of a sample can be adjusted in a microwell or the samplecan be transferred into the next well under controlling, in case ofhandling samples the position of which should be adjusted in the well orwhich should be transferred into the next well. To further ensure thecontrol and migration, the top end of a tube formed in the well forholding the sample is located upper than the top ends of tubes formed inother wells.

[0098] To enable the migration of a sample among a plural number ofwells, the wells are usually connected to each other via, for example,thin pipes, narrow gaps, thin grooves, filers, resin-filled columns orchannels. The present invention relates to an apparatus wherein a pluralnumber of wells are connected to each other via such a structure havingresistance to a fluid flow.

[0099] Now, illustration will be made on the application the presentinvention to an apparatus wherein a plural number of wells are connectedto each other each via a channel, for example, an apparatus fordetecting chemotaxis of cells or separating chemotactic cells. However,it is obvious from the description given above that the presentinvention is not restricted to apparatuses for detecting chemotaxis ofcells or separating chemotactic cells but applicable to variousapparatuses.

[0100] In the apparatus for detecting chemotaxis of cells or separatingchemotactic cells, a cell suspension is put into one of the wells whilea specimen solution is put into the other well. Then it is detectedwhether or not cells migrate toward the well holding the specimensolution, or cells which have migrated are selectively collected. Inthis apparatus, for example, the well holding the cell suspension isconnected to the well holding the specimen solution via a channel. Thus,the state where the cells are passing through the channel is observed,or the cells which are passing or have passed through the channel arecounted.

[0101] A channel which makes it possible to observe or detect thepassage of individual cells has resistance to fluids. In an apparatusprovided with such channels, it is sufficient to employ only a smallamount of cells as a sample, which brings about a merit of beingadequate for examining rare cells. In addition, there is another meritthat quantitative analysis can be made. In this case, however, the wholeapparatus is in a small size and thus samples should be handled inmicroquantities. As a result, there frequently arises unexpectedmigration of cells toward a well holding a specimen solution under theeffect of an increase in pressure caused by the injection into thewells. In case the wells are not held horizontally after the injection,moreover, cells would migrate. These unexpected migrations of cellsresult in confusion in the judgment whether the specimen is achemotactic factor or not. To accurately detect the migration of cellstoward a well holding a specimen solution by their own actions, it istherefore required to prevent the migration of the cells at the point ofinjecting a sample or after the injection.

[0102] As one of countermeasures thereto, the present inventors haveproposed a structure wherein each well has a tube for injecting a sampleand an additional well connected thereto for relieving an increase inpressure at the injection (Japanese Patent Application No. 2001-226466).Now, this structure will be briefly described by reference to FIGS. 1and 2.

[0103] In the apparatus shown by FIG. 1, a cell suspension is injectedinto a well 2A through a tube 3A. A specimen solution is injected into awell 2B through a tube 3B. In case where this specimen contains achemotactic factor, cells tend to migrate from the well 2A toward thewell 2B and thus pass through the channel 1. In FIG. 1, a gapcorresponding to the cell size is provided between a bank 8 formed on asubstrate 5 and a transparent glass substrate 6. Alternatively, barriersconstituting a plural number of thin grooves through which individualcells can pass may be formed. The state of the cells passing through thechannel 1 can be observed by, for example, a microscope 9 through theglass substrate 6. FIG. 2 is a bottom plan view of the substrate 5.

[0104] In the apparatus shown by FIGS. 1 and 2, the tubes 3A and 4A andthe tubes 3B and 4B are connected to each other in the respective wells.In this structure, pressure is dispersed through the tubes connected toeach other. In the present invention, in contrast thereto, the top endsof all of the tubes formed in respective wells have a space in common inwhich a liquid can be held. Owing to this structure, the migration atthe injection can be more surely relieved or the migration can becontrolled (see FIGS. 3 and 4).

[0105]FIG. 3 shows an example of the structure according to the presentinvention which is a unit consisting of a substrate 5, a block 7 and aglass substrate 6. In FIG. 3, a space 10 is held in common by the topends 3Ab and 3Bb of tubes 3A and 3B formed in respective wells. Thewhole apparatus is filled up with a liquid not affecting chemotaxis suchas a buffer solution. The amount of the liquid is sufficient for atleast filling up a part of the space 10. Owing to this liquid, the wholeapparatus is maintained under a definite pressure. Moreover, theresistance of the liquid contributes to the prevention of rapidmigration of a sample caused by the injection pressure and horizontaloff balance of the wells. FIG. 4 shows another example of the structureaccording to the present invention. In this unit, wells are providedwith tubes 3A and 3B for injecting a sample and, further, tubes 4A and4B connected thereto and a space 10 is provided by the top ends 3Ab,4Ab, 3Bb and 4Bb of all of these tubes in common.

[0106] In the step of collecting the migrated cells by sucking from awell holding the specimen through a tube formed in the well, the innerpressure is reduced and thus the samples in wells are mixed each other.In the structure as shown by FIG. 4, this phenomenon can be particularlyeffectively relieved.

[0107] In case of detecting chemotaxis of cells or separating cells, itis preferable that the injected cells are first brought together in thevicinity of a channel in a well. In case of the apparatus for detectingchemotaxis of cells or separating chemotactic cells as shown by FIG. 3,for example, it is preferable that cells injected into the well 2Athrough the tube 3A are located in the vicinity of the channel 1.Namely, these cells may be considered as an example of a sample whoseposition in a well should be adjusted. This position adjustment can becarried out by sucking an appropriate amount of the liquid at anappropriate speed from the well 2B located oppositely across the channelthrough the tube 3B. The amount of the liquid to be sucked is determinedbased on the capacities of the tube and the well after discharging theliquid from the space 10. The amount of the liquid to be sucked and thesucking speed can be easily controlled by a computerized program.

[0108] The present invention further involves in its scope, as amodification of the above-described structure, a microsample treatmentapparatus such as an apparatus for detecting chemotaxis of cells havinga structure wherein the top end of a tube formed in a well for holding,for example, a cell suspension is located upper than the top end of atube formed in another well opposite thereto across a channel (see FIGS.5 to 7). In FIG. 5, a block 7 having a tube mounted thereon has been cutdownward around the top end 3Bb of a tube 3B formed in a well 2B. Thus,the top end 3Ab of the tube 3A in a well 2A is located upper than thetop end 3Bb of the tube 3B. At first, the amount of a liquid filling upthe whole apparatus is controlled so that the liquid level is locatedabove the top end 3Ab of the tube 3A, i.e., the position indicated bythe arrow I in the figure. When cells are injected into the well 2Athrough the tube 3A in this state, rapid migration of the cell isprevented due to the uniform pressure in the whole apparatus and theresistance of the liquid. Thus, the cells scatter in the tube 3A and thewell 2A. Next, the liquid is sucked off from the space 10 so that theliquid level is lowered to the position indicated by the arrow II (i.e.,such a level as making the tope end 3Ab of the tube 3A visible above theliquid face3A). Further, an appropriate amount of the liquid is suckedoff and thus the cells scattering in the vicinity of the channel in thewell 2A can be brought together. The amount of the liquid to be suckedoff can be calculated based on the capacities of the tube 3A and thewell 2A. In usual, the object can be achieved by sucking off the liquidin an amount {fraction (1/10)} to ⅓ times as much as the capacities. Byinjecting the specimen solution into the well 2B after returning theliquid level to the position indicated by the arrow I, a rapid change inpressure at the injection can be relieved.

[0109] As the liquid employed for returning the liquid level to theposition indicated by the arrow I, it is preferable to use a liquidhaving a lower specific gravity than the liquid preliminarily containedin the apparatus (e.g., an aqueous solution such as a buffer solution).Thus, the upper part of the tubes in each well can be covered with theliquid having the lower specific gravity and thus the unnecessarydiffusion of the sample can be prevented owing to the covering effect.An arbitrary liquid can be selected therefor so long as it is inert tothe sample, insoluble in water and has a specific gravity lower than1.0. Examples thereof include Mineral Water M3516 (specific gravity:0.84, manufactured by Sigma) and liquid paraffin.

[0110]FIG. 6 shows an example of a unit having tubes 3A and 3B forinjecting a sample and tubes 4A and 4B connected thereto in each well,wherein the top ends 3Ab and 4Ab of the tubes in a well 2A are locatedupper than the top ends 3Bb and 4Bb of the tubes in another well 2B.FIG. 7 shows an example wherein a slope is formed on a block 7 so thatthe top ends of tubes in a well 2A are located upper. These figures showthe examples wherein the top ends of some tubes are located upper thanthe top ends of other tubes. Various modifications can be further madeto achieve the same object.

[0111] The above-described structure wherein the top ends of some tubesare located upper than the top ends of other tubes is effective in theconnecting manners as will be described hereinbelow. If necessary, otherunit(s) may be further jointed and connected to a double system forconnecting wells each via a channel as shown by FIGS. 3 to 7 to therebygive, for example, a triple system shown by FIG. 8. In FIG. 8, forexample, cells are put into a well 2A, a chemotactic factor is put intoa well 2C and a specimen solution is put into a well 2B. Thus, it can beexamined whether or not the specimen solution inhibits the chemotacticfactor. Moreover, multiple systems are applicable to various purposes.

[0112] As FIG. 9 shows, it is also possible to construct a so-calledconcentric system wherein a plural number of wells are connected to eachother each via a channel around a single well. Furthermore, a concentriccircular system as shown by FIG. 11 may be constructed as a modificationof the type of FIG. 9. Although a triple system is employed in theexample of FIG. 11, it is also possible to employ a double system. Inthe example of FIG. 9, a tube 3A is mounted to a penetrating hole 3Aa.Similarly, tubes 3B₁₋₄ are mounted to penetrating holes 3B_(1a-4a) whiletubes 4B₁₋₄ are mounted to penetrating holes 4B_(1a-4a) respectively. Acell suspension is supplied into a well 2A through the tube 3A andvarious specimens are supplied into wells 2B₁₋₄. Thus, a plural numberof chemotactic factors can be examined at the same time. By supplying asample containing a plural types of cells, the cells can be separateddepending on types at once (i.e., sorting). For example, chemotacticfactors corresponding to respective cell types are put into the wells2B₁₋₄ and a sample containing plural types of cells (for example, wholeblood) is supplied into the central well 2A. Then the cells contained inthe sample migrate toward the wells 2B₁₋₄ containing the correspondingchemotactic factors. After a definite period of time, the cells arecollected from each of the wells 2B₁₋₄ through the tubes 3B₁₋₄ or cellshaving migrated into the wells 2B₁₋₄ are identified.

[0113] In the well-connecting manners as shown by FIGS. 8, 9 and 11, thetubes 3 and 4 are connected to each other in the well 2 in which theyare provided. In these connecting manners, all of the tubes hold at thetop ends thereof a space 10 in common. The top ends of the tubes in awell into which cells are injected are located upper than the top endsof other tubes. Then a liquid is supplied so that the top ends of thetubes in the well into which cells are injected are submerged (see FIG.10). FIG. 10 is a sectional view of the apparatus shown by FIG. 9 alongthe dashed and dotted line. In this example, the top ends 3Ab and 4Ab oftubes 3A and 4A in a well 2A are located upper than the top ends3B_(1b-4b) of tubes 3B₁₋₄ in other wells 2B₁₋₄. An arrow I shows thatthe level of the liquid filling up the space 10 is located above the topends 3Ab and 4Ab of the tubes 3A and 4A. Cells injected into the well 2Athrough the tube 3A scatter in the tube 3A and the well 2A. Then theliquid in the space 10 is sucked off and thus the liquid level islowered to the position indicated by another arrow II so that the topend 3Ab of the tube 3A becomes visible above the liquid face. Then anappropriate amount of the liquid is further sucked off. Thus, the cellsin the well 2A can migrate toward the wells 2B₁₋₄ and thus are broughttogether in the vicinity of the channel 1 toward respective wells. Theamount of the liquid to be sucked off can be calculated based on thecapacities of the tube 3A and the well 2A. Thus, the chemotaxis of thecells in the well 2A concerning the wells 2B₁₋₄ can be examined underthe same positional conditions.

[0114] As another example of the embodiment to which the structure ofthe present invention can be applied, an apparatus shown by FIG. 21 maybe cited. Namely, FIG. 21 is a model view of an apparatus wherein areaction is carried out in a well 2A followed by a treatment through acolumn 16 and then unadsorbed substances passing through the column arecollected from a well 2B. In this case, the column serves as an obstaclehaving resistance to fluids. Substances to be reacted are put into thewell 2A in the state that the liquid level is at the position indicatedby an arrow I. After the completion of the reaction, the liquid level islowered to the position indicated by another arrow II. After furthersucking, the reaction mixture migrates from the well 2A toward thecolumn 16. By further sucking, a substance passing through the columnmigrates toward the well 2B. In case where the substance adsorbed by thecolumn is the target substance, the eluate is supplied into the columnvia the well 2A. Thus, the eluted substance can be collected in the well2B.

[0115] In addition to the above-described case, various applications canbe made. That is to say, interactions among substances can be examinedat the level of microquantities by controlling the migration of samplesamong wells which are connected to each other. For example, theseapparatuses are applicable to antigen/antibody reactions,enzyme/substrate reactions, reactions between soluble receptors andligands, and so on.

[0116] In the well 2A of the apparatus shown by FIG. 5, for example, anantibody bonded to plastic beads of a definite size is reacted with anantigen protein. Then the liquid level is lowered from I to II andfurther the liquid is sucked off. Thus the unreacted antigen proteinpasses through the groove in the barrier 12 and migrates into the well2B. However, an antigen which has reacted with the antibody bonded tothe plastic beads cannot pass through the groove in the barrier 12because of the presence of the beads, thereby being separated from theunreacted antigen protein. Thus, substances can be separated byappropriately selecting the combination of the bead size and the groovewidth.

[0117] It is also possible to use magnetic beads. For example, magneticbeads having a uniform particle size, which are composed of polymercores having a magnetizable substance (for example, γFe₂O₃, Fe₃O₄)uniformly distributed therein and a hydrophilic polymer coating, arecommercially available (Dynabeads® manufactured by DYNAL, Norway) . Bybonding various antibodies onto the surface of these beads, the magneticbeads can be bonded to cells or proteins. By bringing close to apowerful magnet (MPC), the magnetic beads are magnetized and attractedto the magnet. When the magnet is moved away, the beads are demagnetizedand thus scatter again. These characteristics have been used inpurifying cells, proteins, etc. For example, Kanegasaki, S. et al.isolated peripheral B lymphocytes by using magnetic polystyrene beads(manufactured by DYNAL) coated with CD19 antibody (J. Biochem.,117:758-765 (1995)).

[0118] In an apparatus shown by FIG. 22, a protein mixture andantibodies labeled with magnetic beads (magnetic antibody beads) areinjected into a well 2A at the liquid level I. After adsorbing a proteinreacted with the antibody by a magnet 24 provided at the bottom of thewell 2A, the liquid level is lowered to II. Then the liquid is suckedoff from a space 10. Thus, a protein unadsorbed by the magnet 24 alonemigrates into another well 2B. By selecting an appropriate antibody, adesired protein can be thus separated or an unnecessary protein can bethus eliminated. For separate proteins with the use of magneticmaterials, it has been a practice to use columns. However, treatmentswith columns can be performed on the milliliter scale and, therefore,are unsuitable for treating proteins in microquantities. By using theapparatus according to the present invention, proteins can be separatedeven on the scale of several microliters or less.

[0119] The present invention makes it possible to downsize the wholeapparatus and thus samples can be treated in microquantities. Moreover,it is possible to integrate multiplicity of units and thus a largenumber of specimens can be treated at the same time. In addition, thetreatment can be easily automated by programmed control of suction andinjection of liquids.

[0120] That is to say, the apparatus can be automated by providing aunit part having a single unit, an integration unit having a pluralnumber of units of the same or different types or a plural number ofintegration units, liquid level control pipette(s) and a system forcontrolling the movements of the liquid level control pipette(s). Theoperations of the liquid level control pipette(s) are controlled asfollows. Namely, a definite amount of a liquid, which is contained inthe space held by the top ends of a plural number of tubes in common ineach unit, is sucked by the liquid level control pipette(s) to therebyadjust the position of a sample in the well, or transfer the sample intothe next well and, if necessary, the liquid in the compensatory amountis supplied from the liquid level control pipette(s) into the space tothereby return the liquid face to the original level. These controllingoperations can be easily carried out by computerized programming.

[0121] It is also possible to automate the whole apparatus involving thesteps of supplying and collecting a sample, a specimen, a reagent, etc.by providing a unit part, a sample reservoir, a specimen reservoir andsample supply pipette(s) and specimen supply pipette(s) movable overthese parts and further a system for controlling the operations of thesepipettes. If necessary, it is also possible to add a pipette washingpart and a system for controlling the operation of washing the pipettesin the pipette washing part.

[0122] Next, the structure of the apparatus according to the presentinvention will be described in greater detail by reference to anapparatus for detecting chemotaxis of cells as an example. However, itis to be understood that the present invention is not restricted to anapparatus for detecting chemotaxis of cells but applicable to otherapparatuses in order to solve similar technical problems as discussedabove.

[0123] 1) Structure of Unit

[0124] As FIG. 3 shows, a channel 1 and wells 2A and 2B are integrallyformed on a substrate 5. The substrate 5 has holes (penetrating holes)3Aa and 3Ba for mounting tubes 3A and 3B connected to respective wells.A block 7 having the tubes 3A and 3B is fixed so as to fit for thepenetrating holes 3Aa and 3Ba. In the upper part of the block, a space10 commonly held by the top ends 3Ab and 3Bb of the tubes 3Aand 3B isprovided. The bottom face of the substrate 5 is adhered to an opticallypolished glass substrate 6. The block 7, the substrate 5 and the glasssubstrate 6 may be pressed and fixed by fastening, for example, with anO-ring or a packing (see FIG. 20). Alternatively, the substrate 5 andthe glass substrate 6 may be integrally formed. Alternatively, thesubstrate 5, the glass substrate 6 and the block 7 may be integrallyformed. As FIG. 4 shows, the tubes formed in the wells 2A and 2B may befurther provided with tubes 3A, 3B, etc. for injecting/collecting asample and tubes 4A, 4B, etc. for relieving pressure changes. As FIGS.5, 6. etc. show, the space 10 may be partly cut downward to form aconcave. Alternatively, a slope may be formed as shown by FIGS. 7, etc.

[0125] 2) Well

[0126] Wells 2 are formed for holding a sample (i.e., a cell suspension)or a specimen solution such as a solution containing a chemotacticfactor or a solution containing an inhibitor therefor. The capacity ofthe wells is not particularly restricted, so long as a liquid can beheld therein in the minimum amount needed. For example, it is sufficientthat the depth ranges from about 0.05 to about 0.1 mm, the width isabout 1.2 mm and the length is about 2.5 mm. It is also possible toprovide a wall orthogonal to a channel in one or both of wells connectedto each other via the channel (for example, the well for holding cells)to thereby restrict the amount of the liquid in the vicinity of thechannel. Thus, the position of cells in the well can be adjusted (FIG.24). FIG. 24 shows an example wherein wells 2A and 2B are connected toeach other via a channel 1 and walls 24A and 24B are formed inrespective wells orthogonally to the channel 1. Although the distancebetween the walls 24 and the channel 1 may be arbitrarily determined, itusually ranges from 50 to 300 μm.

[0127]FIG. 25 shows modification examples of the well unit having wallsprovided orthogonally to the channel. That is, FIG. 25(1) shows anexample wherein a channel is formed in a part of the well width; (2)shows an example wherein a channel is halved at the center, a couple ofwells (2B, 2C) are provided opposite to a single well (2A) across thechannel, and a wall 24 is formed exclusively in the well 2A side; and(3) shows an example wherein two arrays of barriers are formed in bothsides of a terrace 11 in a channel. Needless to say, these modificationsare cited merely by way of example and thus the present invention is notrestricted thereto. If necessary, a terrace may be formed between thewall provided orthogonally to the channel and the bank.

[0128] 3) Channel

[0129] Now, an example of the structure of a channel 1 (FIGS. 1, 3 and4) will be illustrated by reference to FIG. 12. The channel 1 is a spaceprovided between a bank 8 (a convex on a substrate 5) partitioning wells2A and 2B at both ends and a glass substrate 6. The bank 8 partitioningthe wells 2A and 2B formed at both ends of the channel 1 is notrestricted in size. For example, the height of the bank 8 may range fromabout 0.03 to about 0.1 mm, while the length in the direction toward theopposite well may range from about 0.01 to about 0.5 mm and the lengthin the direction orthogonal to the direction toward the opposite wellmay be about 1.2 mm.

[0130] In a preferred embodiment, a plural number of barriers 12 areformed on the bank to thereby constitute grooves 13 through which cellpass, as shown by FIGS. 13 to 15. In case where no barrier constitutinggrooves is formed in the upper part of the bank, a terrace providing agap or a depth fit for the diameter or deformability of cells is formedbetween the upper face of the bank and the glass substrate. In thiscase, the depth usually ranges from 3 to 50 μm depending on the type ofcells. That is to say, the width may range from 3 to 10 μm (for example,4, 5, 8 or 10 μm) in case of neutrophils, eosinophils, basophils,monocytes/macrophages, T cells, B cells and the like, and from 8 to 20μm in case of cancer cells and cells existing in tissues.

[0131] By forming flat terraces in both sides of the barriers on theupper face of the bank, the passage of cells can be more easilyobserved. Thus, it is favorable to form terraces 11 (FIG. 12), thoughthey are not essentially required. In case of providing the terraces 11,the length thereof in the direction toward the opposite wellappropriately ranges from about 0.01 mm to about 0.5 mm.

[0132] By forming multistage terraces 11 as FIG. 23 shows, cells putinto wells in one side can be easily brought together in the vicinity ofthe bank 8 by sucking from the other side to adjust the position of thecells in the well. In case where the cells are neutrophils, eosinophils,basophils, etc., for example, the distance between the terraces 11 ⁻²and 11 ⁻³ and a glass substrate 6 (i.e., corresponding to the height ofa barrier 12 in the figure) is set to 3 μm and the distance between theterraces 11 ⁻¹ and 11 ⁻⁴ and a glass substrate 6 is set to 4.5 μm. Thencells are supplied into a well 2A and the liquid is sucked from the sideof another well 2B. Then the cells once stop at the terrace 11 ⁻¹. Next,the cells are liable to bring together between the terrace 11 ⁻² and theglass substrate 6. The distance between each of the terraces 11_(−1 to 4) and the glass substrate 6 can be arbitrarily determineddepending on the sample to be treated. Although these distances usuallyrange from about 3 to 5 μm, the present invention is not restrictedthereto. When the terrace (11 ⁻³) in the side opposite to the wellscontaining the cells is made about 1.5 to 5 times longer than theterrace (11 ⁻²) in the side of the wells containing the cells, the cellshaving passed through the channel can be more easily observed andcounted. Although a barrier 12 is formed in the example shown by FIG.23, the barrier is not always necessary in case where the distancebetween the terraces 11 ⁻² and 11 ⁻³ and the glass substrate 6corresponds to the diameter or deformability of cells.

[0133] In case where barriers 12 (see FIGS. 12 to 14) are formed on theupper face of the bank, grooves 13 constituted by the barriers 12 mayhave an arbitrary cross-sectional shape, for example, a V-shapedsection, a convex section or a semicircular section. It is preferablethat the grooves 13 have a width fit for the diameter or deformabilityof cells. The term “deformability” of cells as used herein means that,in case of flexible cells, the cells can easily change their shape (forexample, into flat or string-shaped cells) owing to the flexibility andthus can pass through a gap having a smaller size than the diameter ofthe cells being in the inherent spherical shape in a free space. Byforming such grooves, cells can be observed at individual level and thusseparated depending on desired types. The width of a groove 13 usuallymay range from 3 to 50 μm. It is preferable that the width allows thepassage of cells one by one. Thus an appropriate width may be selecteddepending on the cell type. The width may range from 3 to 10 μm (forexample, 3, 5, 8 or 10 μm) in case of neutrophils, eosinophils,basophils, monocytes/macrophages, T cells, B cells and the like, andfrom 8 to 20 μm in case of cancer cells and cells existing in tissues.The number of the grooves 13 is determined depending on the width of thebarriers concerning the channel width and the groove width. In casewhere the channel width is 1 mm, the barrier width is 10 μm and thegroove width is 5 μm, for example, the number of grooves is 66 at thelargest. To smoothly perform the detection and observation, the numberof the grooves 13 preferably ranges from 1 to about 100, stillpreferably from about 10 to about 70.

[0134] The length of the barriers 12 ranges from about 5 to about 400μm. For example, use may be made of a barrier length of 5, 15, 20, 30,40, 60, 100, 200, 300 or 400 μm. The width of the barriers 12 per se canbe appropriately determined. In case of employing the structure as willbe shown in FIG. 38 hereinafter, it is effective that the width andlength of the barriers are almost the same.

[0135] As FIG. 15 shows, the grooves 13 constituting the channel 1 maybe connected to each other via one or more grooves 14 orthogonal to thedirection toward the opposite well. Owing to this structure, thediffusion of a substance put into one well toward the other well can beuniformized, or cells under passage can be more accurately understood.In this case, the width of the grooves 13 may be changed stepwise eachtime the grooves intersect grooves 14 orthogonal thereto in thedirection toward the opposite well (see FIGS. 36 and 37). Alternatively,grooves in the direction toward the opposite well may be formed bymutually shifting the positions thereof each time the grooves intersectgrooves orthogonal thereto (see FIG. 38). FIG. 38 shows an examplewherein the grooves are formed as shifting by ½ pitch in the orthogonaldirection. It is also possible that the barriers are jointed to eachother in the direction toward the opposite well (see FIG. 39).Alternatively, arrays of barriers can be formed in two positions in bothside of a terrace which is formed at the center of the bank (see FIGS.25(3) and 40). By using these structures, cells having passed thegrooves can be easily observed and counted. It is desirable that theterrace located at the center has an area which can be included in themicroscopic field. FIG. 40(1) is a top plan view while (2) is asectional view.

[0136] The height of the barrier 12 (i.e., the depth of the grooves) maybe appropriately determined depending on the depth of focus of theobjective lens of a microscope, a CCD camera, etc. to be used inobserving the cell migration. For example, a depth of about 3 to about4.5 μm is preferable in case of an objective lens having a focus depthof 10 to 40× magnification, though the present invention is notrestricted thereto.

[0137] 4) Construction of Well and Channel

[0138] As a material of the substrate 5, it is preferable to usesingle-crystal silicon which can be easily fine processed and isrelatively inert to cells. The barriers 12 and the grooves 13 in thechannel 1 can be constructed by subjecting the single-crystal silicon tophotolithography or etching (for example, wet etching or dry etching)employed in manufacturing integrated circuits. The wells 2 and thepenetrating holes 3 a and 4 a, which are larger than the barriers 12 andthe grooves 13, can be constructed by using various known engineeringtechniques such as sand blasting and dry etching. In addition tosingle-crystal silicon, use can be made of hard glasses, hard plastics,metals, etc., so long as a microstructure can be constructed in thechannel. In case of using plastics, it is preferable to employ atreatment for making the surface hydrophilic, for example, forming ahydrophilic film on the surface. It is also possible to separatelyconstruct the channel 1 and the wells 2 and then combine them together.

[0139] 5) Block and Tube

[0140] As shown by FIG. 3, the block 7 is a member located on thesubstrate 5 and having tubes connected to wells. The tubes usually havea square or circular cross-sectional shape. Although these tubes are notrestricted in size, a square tube has a side length of about 1 mm whilea round tube has a diameter of about 1 mm in usual. To hold a cellsuspension or a specimen solution in a desired volume, it is necessarythat these tubes have a length of about 2 to about 10 mm. The materialsof the block or tubes maybe selected from among glasses, plastics suchas acrylic resins and metals. The tubes can be easily produced by usingcommonly employed engineering techniques such as mechanical drilling orlaser drilling. Similarly, the space held commonly by the top ends ofthe tubes can be formed above the block 7 by usual engineeringtechniques.

[0141] To inject cells or a specimen into each unit by hands (i.e.,manually), the periphery of the top end of each supply tube may be cutdownward to thereby form a funnel-shaped concave. Thus, a pipette can beeasily inserted (29 in FIGS. 35(1) and (2)).

[0142] 6) Glass Substrate

[0143] As shown by FIG. 3, the glass substrate 6 is tightly pressed onthe substrate 5 to provide a space in which a liquid is held, therebyenabling the observation of cells passing through the channels. Thus,the glass substrate 6 should remain optically transparent and flat andprovide a plane to which cells can adhere. Use can be made therefor ofglass as well as plastics such as transparent acrylic resins, so long asthe above objects can be achieved thereby. Although its thickness is notparticularly restricted so long as no strain arises in the step ofpressing onto the substrate, the thickness adequately ranges from 0.7 to2 mm.

[0144] 7) Arrangement of Multiplicity of Units

[0145] By referring a plural number of wells connected to each othereach via a channel as a single unit, a plural number of units may bearranged and integrated on a single substrate. Thus, an apparatuswhereby a large number of specimens can be treated at the same time canbe obtained. Units of the same type may be arranged in parallel or unitsof different types may be arranged. Next, the types of the arrangementand integration will be described by reference to respective figures.However, it is to be understood that the present invention is notconstrued as being restricted thereto and thus various combinations maybe also employed depending on the purpose.

[0146]FIG. 16 shows an example wherein 12 well units each having acouple of wells connected via a channel as shown in FIG. 4 are mountedon a square substrate 7 (16 mm×16 mm). In this example, the units areeach 5.7 mm in the major sides and 1.2 mm in the minor sides and locatedat intervals of 0.8 mm.

[0147]FIG. 17 shows an example of an integration of multiplicity ofintegrated units. In FIG. 17, each of quadrilaterals A₁₋₄, B₁₋₄ and C₁₋₄corresponds to the integration shown by FIG. 16. In this case, thearrays A, B and C are integrations of units of different types.

[0148]FIG. 18 shows an example wherein independent double system unitsare integrated circularly. FIG. 19 is a sectional view of the unit ofFIG. 18 along the dashed and dotted line. Concerning the size, forexample, the width of wells 2A and 2B in the radial direction is 1.5 mm,the width of a channel 1 in the redial direction is 0.5 mm and the widthof grooves13 formed in the channel 1 is 10 μm. In this case, the radiusof the whole unit is 5.0 mm.

[0149]FIG. 26 shows an example wherein 12 units of the type shown byFIG. 24 are integrated.

[0150] In such a case of integrating multiplicity of units, a singleblock 7 and a single glass substrate 6 may be used so as to cover thewhole unit (see FIG. 20).

[0151]FIG. 20 shows an example of the fabrication of an apparatus fordetecting chemotaxis of cells and separating chemotactic cellscomprising multiplicity of units integrated together. A substrate 5having multiplicity of units integrated thereon, a packing 5′ and ablock 7 covering them are placed between a cover cap 17 and anintermediate base 21. A glass substrate 6 is placed between theintermediate base 21 and a bottom base 22 and fastened with screws. Thelocations of the block 7 and the substrate 5 are specified by theintermediate base 21 and fixed by guide pins 20 and guide pin receiverholes 19 provided at the bottom face of the block 7. Alternatively, thesubstrate 5 may be directly pressed and fixed to the block 7.

[0152] In FIG. 20, it is also possible that a substrate 5 having asingle unit (i.e., a couple of wells and a channel) is used as asubstitute for the integrated unit and a plural number of the fabricatedunits are arranged at definite intervals. In this case, units can besuccessively exchanged.

[0153] 8) Automatic Controlling System

[0154] Next, the automatic controlling system in the microsampletreatment apparatus according to the present invention will beillustrated in detail by reference to an apparatus for detectingchemotaxis of cells as an example. However, it is needless to say thatthis illustration is given merely by way of example and variousembodiments may be further employed for achieving the automation.

[0155]FIG. 27 shows an example of the automatic controlling system ofthe apparatus for detecting chemotaxis of cells according to the presentinvention. In FIG. 27, U represents a unit part, C represents a cellreservoir, S represents a specimen reservoir and W represents a pipettewashing part. The line X-X′ shows an example of the flow line of aplural number of specimen supply pipettes (6 in this case) alignedlaterally, while the line Y-Y′ shows an example of the flow line of aplural number of cell supply pipettes aligned laterally. The unit part Uis set at the pipette flow line position and a space provided above thetop ends of each unit is filled up with a liquid. Cells are held in thecell reservoir, while various specimens are held in the specimenreservoir S. Liquid level control pipettes aligned laterally are locatedabove the unit part 4B to 4A and the flow line thereof is indicated by,for example, Z-Z′ in FIG. 28. Each pipette is moved, for example, asfollows, though it is needless to say that the present invention is notrestricted thereto.

[0156] A definite amount of a cell suspension is sucked from the cellreservoir C by a cell supply pipette. Then the pipette moves along theflow line Y-Y′ to the unit part U and supplies the cell suspension intothe well 2A of each unit through a cell supply tube 3A. Subsequently,the cell supply pipette returns to the position C and stops theoperation, or moves to supply the cell suspension to the next unit.Since cells are precipitated owing to the gravity, it is favorable tostir the cell suspension contained in the cell-reserving container 25immediately before collecting the cells by sucking.

[0157] Next, the liquid in the space 10 in each unit is sucked by aliquid level control pipette and thus the liquid level is lowered to theposition II, as FIG. 28 shows. Subsequently, a definite amount of theliquid is further sucked so as to adjust the position of cells in thewell 2A. Then the liquid level control pipette is elevated to the liquidlevel I position or higher and the sucked liquid is discharged at anypoint on the flow line Z-Z, thereby returning the liquid level in thespace 10 to the position I. Subsequently, the liquid level controlpipette is further elevated and stops its operation, or moves on thenext unit.

[0158] Then a definite amount of a specimen is sucked from the specimenreservoir S by a specimen supply pipette. The specimen supply pipettemoves along the flow line X-X′ to the unit part U and supplies thespecimen into the well 2B through a specimen supply tube 3B.Subsequently, the specimen supply pipette moves along the flow line X-X′to the pipette washing part W wherein it is washed by repeatedly suckingand discharging a washing liquor in a washing tank. Then the pipette iselevated above the liquid level in the washing tank and stops itsoperation, or moves to the next unit part U to supply the specimen.

[0159] Next, the unit part U having the cell suspension and the specimenthus supplied moves in the direction indicated by an arrow → in FIG. 27and stops at the position where the channel 1 agrees with the detectionpart. Thus, the conditions of the cells are detected and recorded. Asthe unit part U moves, the next unit part U comes to the position of thepipette flow line and thus the above operations are repeated. It is alsopossible to move the unit part U together with the specimen reservoir S.In this case, the unit part U and the specimen reservoir S movestogether and thus the next unit part U and the next specimen reservoir Scome to the pipette flow line.

[0160] The cell reservoir C is provided with containers for temporarilyholding cells to be supplied into the unit part U. These containers maybe in any shape, so long as they can play the desired role. FIG. 29shows an example of the containers in the cell reservoir C. A pluralnumber of cell containers 25 are provide,d corresponding to thearrangement of each unit and a plural number of the cell supplypipettes. In the example shown by FIG. 29, an inclined injection part 26is formed to facilitate the injection of cells into each container andavoid waste of cells. It is preferable to further provide an inlet part27 so that the cell suspension can be easily introduced into thecontainers without waste. By using this structure, the cell suspensioninjected at an arbitrary point can be supplied into all containers,thereby saving a lot of time and labor for injecting the cell suspensioninto individual cells. It is also preferable that the cell containers 25are tapered at the bottom so as to avoid waste of the cell suspension inthe step of sucking by the pipettes. In FIG. 29, (1) is a perspectiveview; (2) is a top plan view; (3) is a sectional view along the dottedline A-A′ in (2); and (4) is another sectional view along the dottedline B-B′ in (2).

[0161] The specimen reservoir S is provided with containers fortemporarily holding a specimen to be supplied into the unit part U.These containers may be in any shape, so long as they can play thedesired role. In case of supplying many types of specimens into the unitpart U, use is frequently made of a method wherein individual specimensare manually injected into the containers in the specimen reservoir Swith the use of micropipettes, etc. In such a case, it is preferable toprovide pipette tip inlet ports 29 having a diameter larger than thepore size of the opening of the containers, as shown in FIG. 30. It isalso desirable that the containers are tapered at the bottom to lessenthe specimen remaining therein after taking out from the containers, asshown by FIG. 30. In FIG. 30, (1) is a perspective view; (2) is asectional view; and (3) is a top plan view. In the example shown by FIG.30(2), the pipette tip 34 is inserted into the container 28 from thepipette tip inlet port 29 in the step of manually injecting a specimen.FIG. 31 shows an example wherein a plural number of specimen containersare located along the flow line X-X′ of the specimen supply pipette. AsFIG. 31 shows, the inlet ports may be alternately located so that theintervals among the containers can be adjusted fit to the intervalsamong the units in the unit part U. The specimen containers may have asquare shape, as shown by FIG. 32. FIG. 33 shows an example wherein aplural number of specimen containers are located along the flow lineX-X′ of the specimen supply pipettes.

[0162] In the pipettes to be used in the apparatus according to thepresent invention, suction and discharge of liquids can be controlled bycomputerized programming. It is preferable to use a pipette having amultichannel syringe as shown by FIG. 34. The needle (tip) of thepipette may be made of glass, a metal, a plastic material, etc. In FIG.34, (1) is a top plan view; and (2) is a side plan view.

[0163] The detection means to be used in the present invention may beany means so long as cells which are passing through a channel or havepassed therethrough can be detected thereby. If necessary, it involves ameans of recording the detection data. Any means known as a means ofdetecting and recording cells is usable therefor. Use can be made of,for example, a microscope optionally combined with a video camera. It isalso possible to employ a system having an objective lens provided witha CCD camera. For the detection in integrated units, it is preferable toemploy a system wherein the channels of the units are successivelyscanned along with an objective lens.

[0164] As shown by FIG. 4, the detection means is usually provided in achannel of a unit. In an apparatus having multiplicity of unitsintegrated together, it is also possible to employ a system whereinarrays of the units successively come to a detection part located at adefinite position for detection and recording. In this case, thechannels of the aligned units are scanned with the detector. Either oneor more scanning detectors may be employed. Owing to this constitution,a relatively small number of detectors suffice for the detection inmultiplicity of integrated units.

[0165] Cells which are passing or have passed through a channel can bedetected and counted by directly observing the cells with a microscope.Alternatively, the detection and counting can be easily performed bypreliminarily labeling the cells with a luminous or fluorescentsubstance and then capturing the luminescence or fluorescence in aconventional manner.

INDUSTRIAL APPLICABILITY

[0166] According to the structure of the present invention, it ispossible to, in the step of injecting a liquid sample into a well,prevent the migration of the sample into another well or overflowthereof. Moreover, the position of the injected sample can be adjustedin a well or the sample can be transferred into the next well undercontrolling.

[0167] The structure according to the present invention achieves aremarkable technical merit and widely applicable particularly in casesof handling samples in microquantities such as solutions and cellsuspensions, or separating cells or particles depending on size.

[0168] A high technical merit can be established by applying thestructure of the present invention to an apparatus for detectingchemotaxis of cells or an apparatus for separating cells with the use ofcell chemotaxis. That is to say, unexpected migration of a sample causedby pressure changes in the step of injecting/sucking samples such ascells and specimen solutions can be prevented thereby. Furthermore,unexpected migration of a sample caused by horizontal off balance of theapparatus can be prevented. Thus, movements of cells by their ownactions can be accurately understood or desired cells can be taken out.Namely, it is possible to obtain results affected by both of the effectof a chemotactic factor or an inhibitor and the characteristics of thecells.

[0169] In the apparatus for detecting chemotaxis of cells or theapparatus for separating cells with the use of cell chemotaxis accordingto the present invention, a bank is formed in a channel located betweenwells or barriers constituting definite grooves are formed on the bankor, alternatively, a gap is formed between a plane provided on the upperface of the bank and a glass substrate. Owing to this structure, itbecomes possible to easily establish the state wherein cells are broughttogether in the vicinity of the channel and aligned in the flowdirection of the cells, when a cell suspension is put into one well andan adequate amount of a liquid is sucked from the other well. As aresult, the presence/absence of the cell chemotaxis can be accuratelydetected.

[0170] The structure according to the present invention makes itpossible to downsize the apparatus. When applied to an apparatus fordetecting chemotaxis of cells or separating chemotactic cells, namely,samples can be used in an amount {fraction (1/50)} to {fraction(1/1000)} times as much in the conventional cases with the use of aBoyden chamber. That is to say, biological samples (whole blood, etc.)per se are usable as samples in the apparatus of the present invention.By using whole blood as a sample, for example, measurement can be madeby using 0.1 μl of blood in case of detecting the chemotaxis ofneutrophils and about 1 μl of blood in case eosinophils, monocytes orbasophils.

[0171] In the structure according to the present invention, moreover, nodelicate control is needed in the step of injecting a liquid, whichbrings about an additional merit that the apparatus can be easilyautomated.

[0172] The unit of the apparatus according to the present invention canbe in a microsize and thus multiplicity of the units can be integratedtogether, which brings about another merit that an apparatus whereby alarge number of samples can be simultaneously treated can be fabricated.In this case, an apparatus having an automated system of injecting anddetecting liquids can be easily fabricated.

[0173] In integrating multiplicity of units, detection and separationfor different purposes can be simultaneously carried out by combiningand integrating units of different types together. Thus, the treatmentefficiency can be elevated. In case of an apparatus for detectingchemotaxis of cells, for example, the detection of various chemotacticfactors or inhibitors for a single type of cells or the detection of thechemotaxis of different types of cells for a single chemotactic factorcan be carried out at once.

1. a microsample treatment apparatus having a structure wherein a pluralnumber of wells are connected to each other via a part having resistanceto fluids and the wells are each provided with tubes forinjecting/sucking a sample and, if necessary, tubes for relievingpressure changes at the injection/suction, characterized in that thesetubes have a space in common at the top ends thereof in which a liquidcan be held.
 2. The microsample treatment apparatus as claimed in claim1 wherein the part having resistance to fluids is selected from amongone or more thin pipes, narrow gaps, thin grooves, filters, resin-filledcolumns and other structures through which a fluid can be passed butwhich have resistance to fluids.
 3. The microsample treatment apparatusas claimed in claim 1 wherein the top end of a tube formed in a well islocated upper than the top ends of the tubes formed in one or more wellsopposite thereto across the part having resistance to fluids.
 4. Themicrosample treatment apparatus as claimed in claim 1 wherein, in one orboth of wells connected to each other via a channel, a wall is formedorthogonal to the channel to thereby restrict the amount of a liquid inthe vicinity of the channel.
 5. A microsample treatment apparatus whichcomprises a unit part having a single unit selected from the microsampletreatment apparatuses as claimed in claims 1 to 4, an integration unithaving a plural number of units of the same or different types or aplural number of integration units, a pipette (or pipettes) forcontrolling the liquid level in the unit part, and a system forcontrolling the operation of the liquid level control pipette(s).
 6. Themicrosample treatment apparatus as claimed in claim 5 characterized inthat the liquid level control pipette(s) are controlled so as to suck adefinite amount of a liquid contained in the space held in common by aplural number of tubes at the top ends thereof in each of the units inthe unit part thereby adjusting the position of the sample in well(s) ortransferring the sample into the respective next well(s) followed by, ifnecessary, supplying the liquid in a compensatory amount to return theliquid face to the original level.
 7. The microsample treatmentapparatus as claimed in claim 5 characterized by having a samplereservoir, a specimen reservoir and sample supply pipette(s) andspecimen supply pipette(s) which are movable over these parts andfurther having a system for controlling the operations of the samplesupply pipette(s) and the specimen supply pipette(s).
 8. The microsampletreatment apparatus as claimed in claim 7 characterized by having apipette washing part wherein the pipettes are controlled so as to repeatsucking and discharging a washing liquor.
 9. An apparatus for detectingchemotaxis of cells or separating chemotactic cells characterized inthat a plural number of wells are connected to each other via a parthaving resistance to fluids, the wells are each provided with tubes forinjecting/sucking a sample and, if necessary, tubes for relievingpressure changes at the injection/suction, these tubes have a space incommon at the top ends thereof in which a liquid can be held, and thewells are closely adhered to a glass substrate in the opposite side tothe tube side.
 10. The apparatus for detecting chemotaxis of cells orseparating chemotactic cells as claimed in claim 9 characterized in thatthe top end of a tube formed in a well for holding cells is locatedupper than the top ends of the tubes formed in one or more wellsopposite thereto across the channel having resistance to fluids.
 11. Theapparatus for detecting chemotaxis of cells or separating chemotacticcells as claimed in claim 9 characterized in that the channel havingresistance to fluids is a bank and a narrow gap is formed between thebank and the glass substrate.
 12. The apparatus for detecting chemotaxisof cells or separating chemotactic cells as claimed in claim 11characterized in that a terrace is formed in the upper part of the bankin the channel and a gap fit for the diameter or deformability of cellsis formed between the terrace and the glass substrate.
 13. The apparatusfor detecting chemotaxis of cells or separating chemotactic cells asclaimed in claim 11 characterized in that barriers constituting one ormore grooves having a width fit for the diameter or deformability ofcells are formed in the upper part of the bank and, if necessary, aterrace is further formed together with the bank and a gap fit for thediameter or deformability of cells is formed between the terrace and theglass substrate too.
 14. The apparatus for detecting chemotaxis of cellsor separating chemotactic cells as claimed in claim 13 characterized inthat a plural number of grooves in the direction toward the oppositewell in the channel are connected to each other via one or more groovesorthogonal thereto.
 15. The apparatus for detecting chemotaxis of cellsor separating chemotactic cells as claimed in claim 14 characterized inthat the width of a plural number of grooves in the direction toward theopposite well in the channel is changed stepwise each time the groovesintersect one or more grooves orthogonal thereto.
 16. The apparatus fordetecting chemotaxis of cells or separating chemotactic cells as claimedin claim 14 characterized in that a plural number of grooves in thedirection toward the opposite well in the channel are formed by mutuallyshifting the positions thereof each time the grooves intersect one ormore grooves orthogonal thereto.
 17. The apparatus for detectingchemotaxis of cells or separating chemotactic cells as claimed in claim13 characterized in that arrays of the barriers constituting the groovesare formed at two positions in both sides of the terrace formed at thecenter of the bank.
 18. An apparatus for detecting chemotaxis of cellsor separating chemotactic cells as claimed in claim 12 characterized inthat multistage terraces are formed on the bank in the channel so as toform gaps with different depths between the terraces and the glasssubstrate.
 19. The apparatus for detecting chemotaxis of cells orseparating chemotactic cells as claimed in claim 18 characterized inthat barriers constituting one or more grooves having a width fit forthe diameter or deformability of cells are formed on the bank andmultistage terraces are formed on the bank.
 20. The apparatus fordetecting chemotaxis of cells or separating chemotactic cells as claimedin claim 9 characterized in that, in one or both of wells connected toeach other via a channel, a wall is formed orthogonal to the channel tothereby restrict the amount of a liquid in the vicinity of the channel.21. An automated apparatus for detecting chemotaxis of cells orseparating chemotactic cells comprising a unit part having a single unitselected from the apparatuses for detecting chemotaxis of cells orseparating chemotactic cells as claimed in claims 9 to 20, anintegration unit having a plural number of units of the same ordifferent types or a plural number of integration units, a cellreservoir, a specimen reservoir and liquid level control pipette(s),cell supply pipette(s) and a specimen supply pipette(s) which aremovable over these parts, and further having a detection part fordetecting cell migration in the unit part and, if necessary, recordingthe detection data which is integrated with the unit part or formed soas to correspond to a plural number of unit parts, and further having asystem for controlling the movements of the liquid level controlpipette(s), the cell supply pipette(s) and the specimen supplypipette(s) and, if necessary, a system for moving the unit part to thedetection part and the next unit part to the flow line of thepipette(s).
 22. The apparatus for detecting chemotaxis of cells orseparating chemotactic cells as claimed in claim 21 characterized byhaving a pipette washing part wherein the pipettes are controlled so asto repeat sucking and discharging a washing liquor.
 23. The automatedapparatus for detecting chemotaxis of cells or separating chemotacticcells as claimed in claim 22 characterized in that the operations of therespective pipettes are controlled as follows: after optionallystirring, a definite amount a cell suspension is sucked by the cellsupply pipette(s) and supplied into the unit part; then a definiteamount of a liquid, which is contained in the space held by the top endsof a plural number of tubes in common in each unit, is sucked by theliquid level control pipette(s) to thereby adjust the position of thecells in the wells; the liquid in the compensatory amount is suppliedfrom the liquid level control pipette (s) into the space to therebyreturn the liquid face to the original level; then a definite amount ofa specimen is sucked from the specimen reservoir by the specimen supplypipette(s) and supplied into the unit part; then the pipettes movetoward the pipette washing part in which they are washed by repeatedlysucking and discharging the washing liquor.